Re: [ccp4bb] changes in small sections of secondary structure
Dear Mahesh, Instead of bothering with the secondary structure assignments why may try to superpose your structures and then view your RMSD plots(Superpose program in CCP4 can do the trick). You might then be better off focusing on the regions with high RMSD. Regards On Sat, Oct 19, 2013 at 3:20 AM, krish krishna@gmail.com wrote: Hi Mahesh, If you have made these screen shots using pymol, then you really need to be careful before you jump to conclude anything from these secondary structural changes (Matt already mentioned it). Assign secondary structures using DSSP or see the secondary structure restraints in the structure while refining. Manual refining of these areas is really important (I mean careful inspection) and once you think these regions you highlighted have proper restraints, then you can conclude to a CERTAIN extent. I would perform MD simulation runs after energy minimization for each structure for 10 to 15 nanoseconds and see whether these small perturbations (what you have depicted) are really there. These runs with identical parameters should give you clear hints how much your protein in different states is stable with time. Also, you can figure out other structural changes with longer simulation times if at all if your protein is dynamic. Hope this helps! Krish On Fri, Oct 18, 2013 at 2:52 PM, Mahesh Lingaraju mxl1...@psu.edu wrote: Hello experts Attached is an image showing different crystal structures of the same protein in diffrent states (mutant vs substrate bound vs unliganded) and highlighted are little changes in secondary structure. I was wondering how real such small changes are and if they are real could they be enough to perturb the energy potential of the protein significantly. I apologize if this is a naive question as this is clearly not my area of expertise. Thanks for your input Mahesh -- -- स्वस्तिक फुलेरा वरिष्ठ अनुसंधान कर्ता संरचनात्मक जीवविज्ञान प्रयोगशाला डी. एन. ए. फिंगरप्रिंटिंग एवं निदान केंद्र हैदराबाद, ५००, ००१ और राष्ट्रीय कोशिका विज्ञान केंद्र पुणे विश्वविद्यालय परिसर, गणेशखिंड पुना, ४११,००७
[ccp4bb] Post-doctoral position in Structural Infection Biology
Postdoctoral Position in Structural Biology of Bacterial Pathogens A postdoctoral position is available for a highly motivated individual at the Department of Medical Biochemistry Biophysics, Karolinska Institutet, Stockholm, Sweden. The candidate will join on-going multidisciplinary research aimed at the structural and functional characterization of proteins and protein complexes from major pathogens, for instance Mycobacterium tuberculosis and ESKAPE pathogens such as Pseudomonas aeruginosa and Staphylococcus aureus. A particular aim of this project is to identify lead compounds that may provide the basis for the development of anti-bacterial drug candidates. The position is for two years, with the possibility of a one-two year extension. The methods used will include e.g. cloning, protein production, protein crystallography, enzyme kinetics, biophysical methods to analyze protein-protein interactions and fragment-based screening of small-molecule libraries for hit identification. Through collaborations with other research groups, approaches such as protein NMR, gene knock-outs, phenotype characterisation and inhibitor design/improvement using pharmacophore modeling and organic synthesis will be employed. The successful candidate must have a doctoral degree, a strong commitment to science, and should possess good written and oral communication skills. A background in biochemistry, biophysics and/or protein crystallography is an advantage. The laboratory has access to state-of-the-art facilities for structural biology and biochemical research. A protein production and crystallization platform and a broad range of instruments for biophysical characterization of proteins protein complexes are available at the department. For further enquiries please contact Prof Gunter Schneider (phone: +46 (0) 8 5248 7675) or by e-mail (gunter.schnei...@ki.se). Interested candidates are encouraged to apply by sending curriculum vitae, a brief statement describing research experience, scientific interests and motivation and the names and e-mail addresses of at least two referees by e-mail to (gunter.schnei...@ki.se). Deadline for applications: december 15, 2013. --- Prof Gunter Schneider Department of Medical Biochemistry Biophysics Scheelevägen 2 Karolinska Institutet, S-171 77 Stockholm, Sweden Phone: +46 8 5248 7675 , mobile phone: 0733 342 877 FAX: +46-8-32 76 26 e-mail: gunter.schnei...@ki.se Home page: http://phillips.mbb.ki.se/
Re: [ccp4bb] changes in small sections of secondary structure
Dear Mahesh, In addition to RMSD plots, you could try to quantitatively analyse the protein residue flexibility in order to detect backbone conformational transitions by means of the method proposed in: Local Fluctuations and Conformational Transitions in Proteins Rocco Caliandro, Giulia Rossetti, and Paolo Carloni J. Chem. Theory Comput. 2012, 8, 4775−4785 By starting from a set of several models of the same protein, the method allows to detect the hinge points of the protein and the residues where transitions between two different conformational states occur. However, by inspecting your picture, also I am not convinced that they are alterations of the protein, but rather inconsistency in assignment of secondary structure. Danilo
Re: [ccp4bb] changes in small sections of secondary structure
Dear Mahesh, You could also run the CCP4 program CONTACT/ACT (Tadeusz Skarzynski Andrew Leslie/Wojtek Rypniewski Howard Terry) for inter residue contacts and compare the NH-OC bonds in the different structures. Best, D -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Swastik Phulera Sent: 21 October 2013 07:52 To: ccp4bb Subject: Re: [ccp4bb] changes in small sections of secondary structure Dear Mahesh, Instead of bothering with the secondary structure assignments why may try to superpose your structures and then view your RMSD plots(Superpose program in CCP4 can do the trick). You might then be better off focusing on the regions with high RMSD. Regards On Sat, Oct 19, 2013 at 3:20 AM, krish krishna@gmail.com wrote: Hi Mahesh, If you have made these screen shots using pymol, then you really need to be careful before you jump to conclude anything from these secondary structural changes (Matt already mentioned it). Assign secondary structures using DSSP or see the secondary structure restraints in the structure while refining. Manual refining of these areas is really important (I mean careful inspection) and once you think these regions you highlighted have proper restraints, then you can conclude to a CERTAIN extent. I would perform MD simulation runs after energy minimization for each structure for 10 to 15 nanoseconds and see whether these small perturbations (what you have depicted) are really there. These runs with identical parameters should give you clear hints how much your protein in different states is stable with time. Also, you can figure out other structural changes with longer simulation times if at all if your protein is dynamic. Hope this helps! Krish On Fri, Oct 18, 2013 at 2:52 PM, Mahesh Lingaraju mxl1...@psu.edu wrote: Hello experts Attached is an image showing different crystal structures of the same protein in diffrent states (mutant vs substrate bound vs unliganded) and highlighted are little changes in secondary structure. I was wondering how real such small changes are and if they are real could they be enough to perturb the energy potential of the protein significantly. I apologize if this is a naive question as this is clearly not my area of expertise. Thanks for your input Mahesh -- -- स्वस्तिक फुलेरा वरिष्ठ अनुसंधान कर्ता संरचनात्मक जीवविज्ञान प्रयोगशाला डी. एन. ए. फिंगरप्रिंटिंग एवं निदान केंद्र हैदराबाद, ५००, ००१ और राष्ट्रीय कोशिका विज्ञान केंद्र पुणे विश्वविद्यालय परिसर, गणेशखिंड पुना, ४११,००७
Re: [ccp4bb] changes in small sections of secondary structure
Hello experts Thanks for your insights. For one of the structures, it turned out to be a rendering issue by pymol like matt pointed out. For the other, the residues are clearly in a less than ideal position. Even if I see deviation from the RMSD plots, i cannot be sure that the structure were refined ideally at those positions ( those are not my structures, i just have the pdb files from my collaborator). Thanks again, Mahesh P.S from what all of you are saying it sounds like those changes are not real, if I find that they could be Ill let everyone know.
Re: [ccp4bb] Problematic PDBs
As a postscript it might be worth mentioning one problematic ligand that suggested to me a way to correct some of the errors mentioned in this thread R12 is indicated as 9-(4-HYDROXY-2,6-DIMETHYL-PHENYL)-3 in the most recent Coot monomer library. But in the PDB ligand description it is 9-(4-hydroxy-2,3,6-trimethylphenyl)-3,7-dimethylnona-2,4,6,8-tetraenoic acid with an additional carbon C16. To make a long story short this ligand was originally deposited missing this extra methyl goup in 1999 (as part of 3CBS) and then apparently updated in 2011 by the PDB. (the relevant lines in the cif are snip R12 C16 C16 C 0 1 N N N ? ? ? -6.631 1.502 0.990 C16 R12 44 R12 H1 H1 H 0 1 N N N ? ? ? -6.602 1.511 2.080 H1 R12 45 R12 H23 H23 H 0 1 N N N ? ? ? -6.422 2.503 0.613 H23 R12 46 R12 H24 H24 H 0 1 N N N ? ? ? -7.619 1.186 0.656 H24 R12 47 snip with the ? ? ? indicating that refined coordinates were not available at the time of the update. There was initially an explanation line at the end of the cif: snip R12 Other modification 2011-10-25 RCSB CS 'add missing methyl group, re-define bond order based on publication' snip But this has mutated for some reason (premature stop codon?) over the past year to the following. snip R12 Other modification 2011-10-25 RCSB snip Obviously the full correct ligand could not have been incorporated into the PDB entry coordinates without these undergoing a full obsolete - supersede process (somewhat embarrassing perhaps as one author is now a wwPDB PI ;) But it is frustrating for users of the PDB that in such cases easily correctable errors are not actually updated by the authors. Would it not be helpful if there were a mechanism to make and track useful improvements in deposited structures? - Perhaps suggested by members of the community to the authors. These changes could be considered as 'corrigenda' and could be documented and tracked - complete with an explanation of the reasoning behind the change and attributing the motivation and origin of the improvement. This would be a good way for the wider scientific community (who maybe do not read this bulletin board) to access the best current model without the authors suffering the full process of retracting and redepositing their PDB entry. The test for obsoleting would then be the same as for a paper - that the change invalidates a fundamental interpretation of the data. All the best Martyn From: Pavel Afonine pafon...@gmail.com To: CCP4BB@JISCMAIL.AC.UK Sent: Sunday, 20 October 2013, 19:49 Subject: Re: [ccp4bb] Problematic PDBs Hello, just for the sake of completeness: this paper lists a bunch of known pathologies (I would not be surprised if they've been remediated by now): http://www.phenix-online.org/papers/he5476_reprint.pdf Pavel On Thu, Oct 17, 2013 at 6:51 AM, Lucas lucasbleic...@gmail.com wrote: Dear all, I've been lecturing in a structural bioinformatics course where graduate students (always consisting of people without crystallography background to that point) are expected to understand the basics on how x-ray structures are obtained, so that they know what they are using in their bioinformatics projects. Practices include letting them manually build a segment from an excellent map and also using Coot to check problems in not so good structures. I wonder if there's a list of problematic structures somewhere that I could use for that practice? Apart from a few ones I'm aware of because of (bad) publicity, what I usually do is an advanced search on PDB for entries with poor resolution and bound ligands, then checking then manually, hopefully finding some examples of creative map interpretation. But it would be nice to have specific examples for each thing that can go wrong in a PDB construction. Best regards, Lucas
Re: [ccp4bb] changes in small sections of secondary structure
Dear Mahesh, Are all the structures at similar resolution? Definition of secondary structure is, and can be, affected by the level of geometric restraints/constraints used in the refinement process. Tony. --- Dr Antony W Oliver Senior Research Fellow CR-UK DNA Repair Enzymes Group Genome Damage and Stability Centre Science Park Road University of Sussex Falmer, Brighton, BN1 9RQ email: antony.oli...@sussex.ac.uk tel (office): +44 (0)1273 678349 tel (lab): +44 (0)1273 677512 On Oct 21, 2013, at 11:55 AM, Mahesh Lingaraju wrote: Hello experts Thanks for your insights. For one of the structures, it turned out to be a rendering issue by pymol like matt pointed out. For the other, the residues are clearly in a less than ideal position. Even if I see deviation from the RMSD plots, i cannot be sure that the structure were refined ideally at those positions ( those are not my structures, i just have the pdb files from my collaborator). Thanks again, Mahesh P.S from what all of you are saying it sounds like those changes are not real, if I find that they could be Ill let everyone know.
Re: [ccp4bb] changes in small sections of secondary structure
Being of an untrusting disposition, I would ask your collaborator for the reflection data as well as the PDBs - it is always a good idea to look at the maps when there is some unexpected structural feature! - The DB provides an electron density server, if the structures have been deposited, otherwise it is straightforward to generate a map yourself and look at it in coot. Eleanor Dodson On 21 October 2013 13:53, Antony Oliver antony.oli...@sussex.ac.uk wrote: Dear Mahesh, Are all the structures at similar resolution? Definition of secondary structure is, and can be, affected by the level of geometric restraints/constraints used in the refinement process. Tony. --- Dr Antony W Oliver Senior Research Fellow CR-UK DNA Repair Enzymes Group Genome Damage and Stability Centre Science Park Road University of Sussex Falmer, Brighton, BN1 9RQ email: antony.oli...@sussex.ac.uk tel (office): +44 (0)1273 678349 tel (lab): +44 (0)1273 677512 On Oct 21, 2013, at 11:55 AM, Mahesh Lingaraju wrote: Hello experts Thanks for your insights. For one of the structures, it turned out to be a rendering issue by pymol like matt pointed out. For the other, the residues are clearly in a less than ideal position. Even if I see deviation from the RMSD plots, i cannot be sure that the structure were refined ideally at those positions ( those are not my structures, i just have the pdb files from my collaborator). Thanks again, Mahesh P.S from what all of you are saying it sounds like those changes are not real, if I find that they could be Ill let everyone know.
Re: [ccp4bb] AW: [ccp4bb] Molecular Replacement using low sequence identity templates
I dont know about LLG scores - they seem extremely variable depending on the degree of sequence similarity you assign. However when you get an R/Rfree of 40%/47% that is a pretty good sign that at least something is correct. It isnt clear whether that is after you have placed 2 copies of the second component? Anyway - maybe try again with the refined component 2 and look for component 1 again? Eleanor On 18 October 2013 15:51, herman.schreu...@sanofi.com wrote: Dear Jan, There are a few things a would do in this case. The first is to check the processing to make sure the space group is really C2 and, although unlikely, not some other space group. The second thing would be to try to place the first component. From your email it is not clear to me whether or not you were able to place the first component after the second component had been placed. In your case, I would give both components to phaser and ask phaser to first place component 2 and then component 1. It might be that the correct solution gets rejected because of clashes, so I would also try to trim the first component, or to increase the number of allowed clashes in Phaser. Although you expect two copies of your heterodimer, you may have a crystal with a high solvent content and only one dimer in the asymmetric unit. In this case the crystal packing should make sense i.e. continuous crystal contacts in all three dimensions. Best, Herman -Ursprüngliche Nachricht- Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Jan Félix Gesendet: Freitag, 18. Oktober 2013 13:17 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] Molecular Replacement using low sequence identity templates Dear all, I have a question regarding Molecular Replacement using low sequence identity templates. I have a 2.7 Angstrom dataset of a heterodimeric protein-protein complex (space group C 1 2 1, no twinning detected using xtriage). For the first component homologs are available, but for the other the best found template only has 20 % sequence similarity. Strangely, I cannot place the first component directly, but the second component can be placed (after trimming the template with chainsaw) using phaser with a TFZ score of 12 and a LLG of about 1200. Although at least 2 NCS copies of the heterodimer are expected based on the unit cell parameters, only 1 copy of the second component gets placed. If I try to place the first component based on the .sol file of the first MR solution, the TFZ score for the second placement is only about 3.5, but if I then try to place this second MR solution (2 components) as a whole I get a RFZ of 8, TFZ of 16 and a LLG of about 1000. However, none of the MR solutions I obtained seems to refine in PHENIX. Using autobuild does not lower the R/Rfree values which seem to get stuck at an R/Rfree of 0.40/0.47. I have tried trimming off loops, simulated annealing, DEN-refinement, morphing and MR-rosetta, but nothing seems to improve the model.. Also, in every MR solution only half of the asymmetric unit seems to be filled, but phaser fails to place more units..As I am seriously starting to doubt the actual content of the crystals, I tried Nearest Cell to search for similar space group, but without any hits. So here is my question. Is it possible to get TFZ/LLG values this high in C 1 2 1 with a completely incorrect model by chance, or can I assume that this MR solution points out that what I think is in the crystal is actually there? And secondly, as I am a bit stuck here, are there any new strategies I can try to tackle this problem? Off course, experimental phasing is an option, but the crystals grew slowly over e few months and I only had 1 drop with 1 crystal, so reproducing the crystals might be though.. Thanks for any tips and best regards, Jan
Re: [ccp4bb] [ccp4bb] Molecular Replacement using low sequence identity templates
Hi Eleanor, Yes, the initial LLG scores in Phaser are highly dependent on the assigned sequence identity, which is translated into an initial estimate of the effective RMSD of the model. However, the latest versions of Phaser refine the RMSD estimate at the end of the job and, assuming that two runs find the same solution and the refinement manages to converge (which it usually does these days), the LLG at the end should be pretty reproducible regardless of the assigned sequence identity. Best wishes, Randy On 21 Oct 2013, at 14:42, Eleanor Dodson eleanor.dod...@york.ac.uk wrote: I dont know about LLG scores - they seem extremely variable depending on the degree of sequence similarity you assign. However when you get an R/Rfree of 40%/47% that is a pretty good sign that at least something is correct. It isnt clear whether that is after you have placed 2 copies of the second component? Anyway - maybe try again with the refined component 2 and look for component 1 again? Eleanor On 18 October 2013 15:51, herman.schreu...@sanofi.com wrote: Dear Jan, There are a few things a would do in this case. The first is to check the processing to make sure the space group is really C2 and, although unlikely, not some other space group. The second thing would be to try to place the first component. From your email it is not clear to me whether or not you were able to place the first component after the second component had been placed. In your case, I would give both components to phaser and ask phaser to first place component 2 and then component 1. It might be that the correct solution gets rejected because of clashes, so I would also try to trim the first component, or to increase the number of allowed clashes in Phaser. Although you expect two copies of your heterodimer, you may have a crystal with a high solvent content and only one dimer in the asymmetric unit. In this case the crystal packing should make sense i.e. continuous crystal contacts in all three dimensions. Best, Herman -Ursprüngliche Nachricht- Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Jan Félix Gesendet: Freitag, 18. Oktober 2013 13:17 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] Molecular Replacement using low sequence identity templates Dear all, I have a question regarding Molecular Replacement using low sequence identity templates. I have a 2.7 Angstrom dataset of a heterodimeric protein-protein complex (space group C 1 2 1, no twinning detected using xtriage). For the first component homologs are available, but for the other the best found template only has 20 % sequence similarity. Strangely, I cannot place the first component directly, but the second component can be placed (after trimming the template with chainsaw) using phaser with a TFZ score of 12 and a LLG of about 1200. Although at least 2 NCS copies of the heterodimer are expected based on the unit cell parameters, only 1 copy of the second component gets placed. If I try to place the first component based on the .sol file of the first MR solution, the TFZ score for the second placement is only about 3.5, but if I then try to place this second MR solution (2 components) as a whole I get a RFZ of 8, TFZ of 16 and a LLG of about 1000. However, none of the MR solutions I obtained seems to refine in PHENIX. Using autobuild does not lower the R/Rfree values which seem to get stuck at an R/Rfree of 0.40/0.47. I have tried trimming off loops, simulated annealing, DEN-refinement, morphing and MR-rosetta, but nothing seems to improve the model.. Also, in every MR solution only half of the asymmetric unit seems to be filled, but phaser fails to place more units..As I am seriously starting to doubt the actual content of the crystals, I tried Nearest Cell to search for similar space group, but without any hits. So here is my question. Is it possible to get TFZ/LLG values this high in C 1 2 1 with a completely incorrect model by chance, or can I assume that this MR solution points out that what I think is in the crystal is actually there? And secondly, as I am a bit stuck here, are there any new strategies I can try to tackle this problem? Off course, experimental phasing is an option, but the crystals grew slowly over e few months and I only had 1 drop with 1 crystal, so reproducing the crystals might be though.. Thanks for any tips and best regards, Jan -- Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical Research Tel: + 44 1223 336500 Wellcome Trust/MRC Building Fax: + 44 1223 336827 Hills RoadE-mail: rj...@cam.ac.uk Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk
[ccp4bb] Fwd: Post-Doctoral Position in Biophysics at the University of Gothenburg, Sweden
*Post-Doctoral Position in Biophysics at the University of Gothenburg, Sweden *A postdoctoral research positions is available from the beginning of 2014 in the biomolecular dynamics group of Dr. Gergely Katona at the Department of Chemistry and Molecular Biology, University of Gothenburg, Sweden. Research and graduate education at the Department of Chemistry and Molecular biology comprises a wide scientific field from the atomic and molecular levels all the way through cells to intact organisms. Much of the phenomena studied here impact on our natural environment and living systems. In the framework of an ongoing program supported by the Knut and Alice Wallenberg Foundation, the postdoctoral fellow will develop tools for studying diffuse scattering in protein crystals. The postdoctoral research fellow will join an interdisciplinary research team investigating the effect of high frequency radiation on proteins and living organisms. The project is part of a collaboration between groups at Chalmers University of Technology and the Department of Chemistry and Molecular Biology at the University of Gothenburg. A strong technical background is required, including demonstrated experience with analysis and modeling of X-ray scattering processes. Candidates for the postdoctoral position are expected to be proficient in coding/scripting (e.g. python, C/C++, shell scripting) and capable of participating in code development. In-depth understanding of X-ray scattering processes is required. Knowledge of the biological field or experience in laboratory work is an advantage, but not essential. Previous experience with infrared spectroscopy and the Comsol Multiphysics package is an advantage. Good communication skills both oral and written English is a requirement. An achieved doctoral degree is compulsory for a position as postdoctor at University of Gothenburg. The doctoral thesis shall be in a relevant area according to the specific position stated here. The postdoctoral fellowship is primarily aimed at applicants who have obtained their doctoral degree in the last three years. Salary for this position is commensurate with experience. The position is initially available for one year and may be extended upon positive evaluation by the Knut and Alice Wallenberg Foundation. The application should include curriculum vitae, publications, a cover letter explaining your research interests and fit for the position and other documents that you wish to include. Please also include two personal references (name, email and phone number). The application should be sent to Dr. Katona (gergely.kat...@cmb.gu.se). * *-- Gergely Katona, PhD associate professor, docent Department of Chemistry and Molecular Biology, University of Gothenburg Box 462, 40530 Göteborg, Sweden Tel: +46-31-786-3959 / M: +46-70-912-3309 / Fax: +46-31-786-3910 Web: http://www.csb.gu.se/katona, Email: gergely.kat...@cmb.gu.se
Re: [ccp4bb] [ccp4bb] Molecular Replacement using low sequence identity templates
I guess my experience is out of date - please ignore comments on LLG! Eleanor On 21 October 2013 15:40, Randy Read rj...@cam.ac.uk wrote: Hi Eleanor, Yes, the initial LLG scores in Phaser are highly dependent on the assigned sequence identity, which is translated into an initial estimate of the effective RMSD of the model. However, the latest versions of Phaser refine the RMSD estimate at the end of the job and, assuming that two runs find the same solution and the refinement manages to converge (which it usually does these days), the LLG at the end should be pretty reproducible regardless of the assigned sequence identity. Best wishes, Randy On 21 Oct 2013, at 14:42, Eleanor Dodson eleanor.dod...@york.ac.uk wrote: I dont know about LLG scores - they seem extremely variable depending on the degree of sequence similarity you assign. However when you get an R/Rfree of 40%/47% that is a pretty good sign that at least something is correct. It isnt clear whether that is after you have placed 2 copies of the second component? Anyway - maybe try again with the refined component 2 and look for component 1 again? Eleanor On 18 October 2013 15:51, herman.schreu...@sanofi.com wrote: Dear Jan, There are a few things a would do in this case. The first is to check the processing to make sure the space group is really C2 and, although unlikely, not some other space group. The second thing would be to try to place the first component. From your email it is not clear to me whether or not you were able to place the first component after the second component had been placed. In your case, I would give both components to phaser and ask phaser to first place component 2 and then component 1. It might be that the correct solution gets rejected because of clashes, so I would also try to trim the first component, or to increase the number of allowed clashes in Phaser. Although you expect two copies of your heterodimer, you may have a crystal with a high solvent content and only one dimer in the asymmetric unit. In this case the crystal packing should make sense i.e. continuous crystal contacts in all three dimensions. Best, Herman -Ursprüngliche Nachricht- Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Jan Félix Gesendet: Freitag, 18. Oktober 2013 13:17 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] Molecular Replacement using low sequence identity templates Dear all, I have a question regarding Molecular Replacement using low sequence identity templates. I have a 2.7 Angstrom dataset of a heterodimeric protein-protein complex (space group C 1 2 1, no twinning detected using xtriage). For the first component homologs are available, but for the other the best found template only has 20 % sequence similarity. Strangely, I cannot place the first component directly, but the second component can be placed (after trimming the template with chainsaw) using phaser with a TFZ score of 12 and a LLG of about 1200. Although at least 2 NCS copies of the heterodimer are expected based on the unit cell parameters, only 1 copy of the second component gets placed. If I try to place the first component based on the .sol file of the first MR solution, the TFZ score for the second placement is only about 3.5, but if I then try to place this second MR solution (2 components) as a whole I get a RFZ of 8, TFZ of 16 and a LLG of about 1000. However, none of the MR solutions I obtained seems to refine in PHENIX. Using autobuild does not lower the R/Rfree values which seem to get stuck at an R/Rfree of 0.40/0.47. I have tried trimming off loops, simulated annealing, DEN-refinement, morphing and MR-rosetta, but nothing seems to improve the model.. Also, in every MR solution only half of the asymmetric unit seems to be filled, but phaser fails to place more units..As I am seriously starting to doubt the actual content of the crystals, I tried Nearest Cell to search for similar space group, but without any hits. So here is my question. Is it possible to get TFZ/LLG values this high in C 1 2 1 with a completely incorrect model by chance, or can I assume that this MR solution points out that what I think is in the crystal is actually there? And secondly, as I am a bit stuck here, are there any new strategies I can try to tackle this problem? Off course, experimental phasing is an option, but the crystals grew slowly over e few months and I only had 1 drop with 1 crystal, so reproducing the crystals might be though.. Thanks for any tips and best regards, Jan -- Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical Research Tel: + 44 1223 336500 Wellcome Trust/MRC Building Fax: + 44 1223 336827 Hills RoadE-mail: rj...@cam.ac.uk Cambridge CB2 0XY, U.K.
[ccp4bb] The binding between disordered and ordered proteins
Dear All: I have a general question about protein- protein interactions. I have two proteins, A and B. A is a disordered protein while B is a well folded protein. The binding between A and B has been approved by GST-pull down assay previously. The strange thing is I cannot get them bind if protein A were just freshly prepared. However, if I kept these two proteins separately for one or two days at 4 degree and then did the GST-pull down assay again, I can observe very strong interaction between A and B. Protein A doesn't contain any cys residue. I have already test certain chemicals which might affect the interactions, for example, DTT and EDTA. These chemicals seems to have no effect on the binding. Although A is a disordered protein, does it need such long time to find its proper conformation? Do any people have similar experience? Any suggestions are greatly appreciated. Thanks, Dee
Re: [ccp4bb] The binding between disordered and ordered proteins
Dear Dee, Some proteins with chaperone-like activity (perhaps your B?) can only bind to partially folded proteins. Probably A folds to a molten globule structure after 1-2 days. You can check by spectroscopic techniques (ANS or Trp fluorescence, CD). Hope that helps. Cheers, Clement On 10/22/13 11:10 AM, Xiaodi Yu wrote: Dear All: I have a general question about protein- protein interactions. I have two proteins, A and B. A is a disordered protein while B is a well folded protein. The binding between A and B has been approved by GST-pull down assay previously. The strange thing is I cannot get them bind if protein A were just freshly prepared. However, if I kept these two proteins separately for one or two days at 4 degree and then did the GST-pull down assay again, I can observe very strong interaction between A and B. Protein A doesn't contain any cys residue. I have already test certain chemicals which might affect the interactions, for example, DTT and EDTA. These chemicals seems to have no effect on the binding. Although A is a disordered protein, does it need such long time to find its proper conformation? Do any people have similar experience? Any suggestions are greatly appreciated. Thanks, Dee
